Protected tyrosine derivatives, method for the production thereof and use of the same for producing o-(2-[18f]-fluoroethyl)-l-tyrosine

ABSTRACT

The compound O-(2-[ 18 F]fluoroethyl)-L-tyrosine has proven to be particularly suitable for positron emission tomography and has already been tested in clinical practice. Until now, the compound has been prepared according to a relatively laborious method (Wester H. J. et al., J. Nucl. Med. 1999; 40: 205-212). 
     The invention relates to L-tyrosine derivatives of the formula (1) 
                 
 
whereby R 1  represents a suitable protective group for the carboxy group, R 2  a suitable protective group for the amino group and R 3  a suitable leaving group,
         R 1  represents a methylthiomethyl group, a tetrahydrofuranyl group, a diphenylmethyl group, a para-methoxybenzyl group, a piperonyl group or a tert-butyl group, R 2  an alkyl- or an arylalkyl group and R 3  a p-tosyloxy, methanesulfonyloxy, trifluoromethanesulfonyloxy or bromine.       

     The invention also relates to a method for preparing O-(2-[ 18 F]-fluoroethyl)-L-tyrosine from the initial compounds of formula (1) and method for the preparation of these initial compounds.

The invention relates to a method for the preparation ofO-(2-[¹⁸F]fluoroethyl)-L-tyrosine, novel compounds as initial materialfor the preparation of O-(2-[¹⁸F]fluoroethyl)-L-tyrosine and a methodfor the preparation of this initial material.

The diagnosis of brain tumors is performed lately more and more throughthe positron emission tomography (PET)-method.

In the beginning the investigations related to the PET-method wereconcentrated on the investigation of glucose metabolism, using thecompound ¹⁸F-fluorodeoxyglucose (FDG) as a diagnostic. With thislabelled compound the results were not satisfactory in all cases, inparticular since glucose is accumulating in the whole brain and for thisreason no sufficient contrast can be observed between normal tissue andtumor tissue and henceforth a satisfactory distinction of tumor tissuefrom healthy brain tissue is not possible.

More satisfactory were the experiences with labelled amino acids.Promising results were achieved initially with the amino acid¹¹C-methionine. These results proved that reliable investigations can beperformed for the determination of brain tumors and the course of atherapy. At first it was assumed that an increased protein synthesis inthe tumor tissue is the cause of the increased amino acid concentration,but nowadays it is assumed that the reason is a change of the amino acidtransport. Such a change of the amino acid transport can be investigatednot only with the normal physiologic amino acids, but also with aminoacid derivatives which themselves cannot be used for protein synthesis.

The investigations with ¹¹C-methionine displayed however the practicaldisadvantage that the half-life of ¹¹C of 20 minutes is very short andthe PET investigations with ¹¹C-methionine (and of course with other¹¹C-labelled compounds) can only be performed in or in the immediatevicinity of an institute where the short-living positron emitter ¹¹C canbe prepared.

To overcome the disadvantage of the short half-life of ¹¹C, furtherinvestigations were performed with 18F-labelled amino acids. Thehalf-life of ¹⁸F is with 110 minutes much more favorable compared with¹¹C. Such a half-life allows a preparation in a central facility withsubsequent transport to other institutions and doctor's surgeries. Inthe meantime, experiences with 4-[¹⁸F]-fluoro-L-proline,O-(2-[¹⁸F]fluoroethyl)-L-tyrosine 1-amino-3-[¹⁸F]fluorocyclobutanecarboxylic acid and 3-[¹⁸F]fluoro-α-methyl-L-tyrosine are available. Thecompound O-(2-[¹⁸F]fluoroethyl)-L-tyrosine was found out to be highlysuitable and is already tested in the clinical practice (Weber W. A. etal.; Eur. J. Nucl. Med. 2000; 27: 542-549).

The preparation of O-(2-[¹⁸F]fluoroethyl)-L-tyrosine used to beperformed with a relatively laborious method (Wester H. J. et al.; J.Nucl. Med. 1999; 40: 205-212).

This method comprised the preparation in a first step of[¹⁸F]fluoroethyl tosylate, and in a second step unprotected L-tyrosine(as the di-potassium salt) is reacted herewith.

A disadvantage of this preparation is the need to purify bychromatography the ¹⁸F-fluorinating reagent before further reaction.

It was an object of the present invention to provide a less laboriouspreparation of O-(2-[¹⁸F]fluoroethyl)-L-tyrosine.

It was a further object of the present invention to provide initialmaterial for the preparation of O-(2-[¹⁸F]fluoroethyl)-L-tyrosine.

It was yet a further object of the present invention to provide a way ofpreparing the initial material.

The invention relates initially to novel initial material for thepreparation of O-(2-[¹⁸F]-fluoroethyl-L-tyrosine ([¹⁸F] FET) accordingto the formula (1):

whereby R¹ is a suitable protective group for the carboxy group and R²is a suitable protective group for the amino group.

The group R³ is suitable leaving group for the fluorination of theinitial material of the formula (1) to the corresponding fluorinatedcompound.

Suitable groups R¹ are ones which protect the carboxy group under theparticular reaction circumstances. As will be described later in moredetail in connection with the description of the method of theinvention, it is a preferred embodiment to perform the method in adipolar aprotic solvent. The group R¹ shall have the function, in thispreferred embodiment of the method, not to be split off under theconditions of the nucleophilic ¹⁸F-fluorination. It is wanted, that thegroup R¹ can be split off easily under acid conditions in a last step ofthe method together with the group R².

Such suitable groups R¹ are for instance such rests, which can also besplit off under strongly acid conditions in an organic, non-aqueoussolvent.

Preferred groups R¹ according to the invention are the methylthiomethylgroup, the tetrahydrofuranyl group, the diphenylmethyl group, thepara-methoxybenzyl group, the piperonyl group and the tert-butyl group.

Most preferred for the group R¹ is the tert-butyl group.

Suitable groups R² are such ones which protect the amino group of theamino acid under the particular reaction circumstances. As will bedescribed in more detail later in connection with the method of theinvention it is a preferred embodiment to perform the method in adipolar aprotic solvent. The group R² shall in this preferred embodimenthave the function of providing the protective effect specifically underthe conditions of the nucleophilic ¹⁸F-fluorination and of preventing aracemisation of the L-amino acid. It is desired that the group can besplit of easily in a last step of the method.

Such groups are for instance alkyl- or arylalkyl groups.

Preferred groups R² according to the invention are carbamates such astert-butylcarbamate, p-methoxybenzylcarbamate, diphenylmethylcarbamateand the triphenylmethyl group. Most preferred is the triphenylmethylgroup.

The group R³ represents a suitable leaving group. This group issubstituted in the actual fluorination method through fluorine-18.

Preferred groups R³ in the scope of the invention are p-tosyloxy,methanesulfonyloxy, trifluoromethanesulfonyloxy and bromine.

A particularly preferred group R³ is the p-tosyloxy group.

It has turned out that the compounds of the formula (1) should bepresent in crystalline form, which makes their handling and dosingsubstantially more easy.

The compounds of the formula (1) are in particular suited as initialmaterial for the preparation of O-(2-[¹⁸F]fluoroethyl)-L-tyrosine.

The method of the invention for the preparation ofO-(2-[¹⁸F]fluoroethyl)-L-tyrosine provides ¹⁸F-fluorinating an initialcompound of the formula (1) in a first step and splitting off theprotective groups R¹ and R² in a second step, thereby obtaining thedesired final compound O-(2-[¹⁸F]fluoroethyl)-L-tyrosine.

For the fluorination a compound of the formula (1) is reacted in thepresence of a phase transfer system, for instance of the formulaR₄N⁺¹⁸F, whereby R represents an alkyl group with 3 to 6 carbon atoms,preferably n-butyl. ¹⁸F is prepared through a method which is known inthe art, by the conversion of water enriched with ¹⁸O through a(p,n)-nuclear reaction in a cyclotron into [¹⁸F]fluoride.

The fluorination is performed with the aid of a phase transfer catalyst.As such phase transfer catalysts in principle catalysts such[K⊂2.2.2]₂CO₃ and (TBA⁺HCO₃*) come into consideration.

It has been shown that a yield of approximately 10% can be achieved whenthe fluorination step is performed with [K⊂2.2.2]₂CO₃ as the catalyst.

Furthermore, it has been shown, that this result can be improved byperforming the fluorination step with (TBA⁺HCO₃ ⁻) as the catalyst. Whenthe particularly preferred initial compoundN-trityl-O-(2-tosyloxyethyl)-L-tyrosine tert-butyl ester is used as theinitial compound for the preparation ofO-(2-[¹⁸F]fluoroethyl)-L-tyrosine, a radiochemical yield of more than80% is achieved with (TBA⁺HCO₃*) as the catalyst. This represents asurprisingly good result.

As the solvent for the nucleophilic ¹⁸F-fluorination the usual organicsolvents come into consideration. It has been shown that the reactionproceeds particularly well with an aprotic solvent such as for instanceacetonitrile, N,N-dimethyl acetamide, N,N-dimethyl formamide, dimethylsulfoxide and hexamethylphosphoric triamide, whereby acetonitrile isparticularly suited due to its physico-chemical properties.

The first step is usually performed at a temperature of approximately85° C.

A duration of 5 minutes is usually sufficient for the performance.

After termination of the first step, as mentioned before, afterapproximately 5 minutes the deprotection is performed. It is notnecessary to perform a prior purification and the circumstance that thesecond step of the deprotection proceeds in the same container as thefirst step, and hence the method relates to a one-pot reaction, is anattractive aspect of the present invention.

The deprotection itself is initiated by the addition of a strong acid,this should be an acid which can also be used in a non-aqueous systemsuch as for instance dichloromethane. The deprotection proceedsparticularly well with trifluoroacetic acid.

After the deprotection the compound O-(2-[¹⁸F]fluoroethyl)-L-tyrosine isavailable in unpurified form.

The last purification step of the final compoundO-(2-[¹⁸F]fluoroethyl)-L-tyrosine is performed with usual tools andmethods. The separation of the labelled amino acid can be performed veryfavorably through solid-phase extraction from the organic phase. The useof silica gel and aluminum oxide is recommendable in this connection.When this extraction is performed with silica gel and aluminum oxide,acid which is usually adsorbed at the solid phase is largely removedthrough a suitable elution step. It is not particularly important whatsolvent is taken for the elution, but for the selection of the solventit plays a role what acid was taken and what solid phase combination wasused. In general it has turned out, that a mixture of n-pentane anddiethylether is very suitable for this elution step in particular whentrifluoroacetic acid is used.

After the elution the remaining solvent rest which is adhering to thesolid phase can be evaporated and removed with an inert gas such asnitrogen or argon and the labelled amino acid can be extracted with asuitable buffer solution. Usually such a buffer solution is used whichallows an immediate HPL-chromatography.

It is the major advantage of the method of the invention, that thelabelled amino acid can be prepared with relative ease in a one-potreaction. The method allows the preparation while avoiding racemisation.The use of [¹⁸F]fluoride guarantees a no-carrier added final product,which is required for an administration to patients. It is the advantageof the method of the invention, that a preparation of a labelled aminoacid with a molar activity of more than 18,5 GBq/μmol is possiblewithout undue efforts.

The preparation of the initial compounds of the formula (1) can beperformed with methods which are known in the art. It is possible in thepreparation to use the amino acid L-tyrosine as the basis and to selectthe further reactants depending on the wanted final product.

When an initial compound of the formula (1) which comprises as the groupR¹ a tert-butyl group needs to be prepared, it is convenient to startwith the compound L-tyrosine tert-butyl ester, since this compound isavailable commercially. Otherwise L-tyrosine is combined in a first stepwith a reagent which is capable of reacting with the carboxy group,thereby forming a compound of the formula (2):

Hereby R¹ has the same meaning as described in connection with thedescription of the initial compounds according to the invention.Suitable reagents for the performance of the reaction are in particularalcohols such as for instance tert-butanol or the olefin isobutene.

In a second step the compound of the formula (2) reacts with a compoundR²X whereby a compound of the formula (3)

is formed. The group R² has hereby the same meaning as described inconnection with the description of the initial compounds according tothe invention. X represents a halogen, preferably chlorine.

In a next step the compound of the formula (3) is converted into acompound of the formula (1), whereby in this last step a R³—C₂H₄— groupis introduced at the hydroxyl group of the aromatic ring. Suitablereagents for this last step are 1,2-bifunctional ethylene derivatives,such as for instance ethylene glycol di (p-toluolsulfonate) and2-bromoethanol.

It is a preferred embodiment of the compounds of the formula (1), thatthe group R³ is a tosyloxy group, for this preferred embodiment the laststep is performed with ethylene glycol di (p-toluolsulfonate).

The invention is now described in more detail on the basis of thefollowing examples:

EXAMPLE 1 Preparation ofO-(2-tosyloxyethyl)-N-(triphenylmethyl)-L-tyrosine tert-butyl ester

First Phase N-triphenylmethyl-L-tyrosine tert-butyl ester

L-Tyrosine tert-butyl ester* was reacted at room temperature in DMF, inthe presence of triethylamine, with an equimolar amount oftriphenylmethylchloride. Through mixing of the DMF phase with the 4-foldamount of ice the raw product is precipitated and recrystallized fromethanol.

Yield: 70% Fp. 171° C.

(*Commercially Available)

Second Phase O-(2-Tosyloxyethyl)-N-(triphenylmethyl)-L-tyrosinetert-butyl ester:

Equimolar amounts of N-triphenylmethyl-L-tyrosine tert-butyl ester andethylene glycol di-(p-toluolsulfonate) were dissolved in acetone. Theeduct mixture was stirred intensely for two days at room temperature.After filtration and evaporation of the solution containing the rawproduct, the purification of the product was performed through columnchromatography on silica gel with an elution agent (?Laufmittel?)consisting of low boiling petroleum ether and ethyl acetate.

Yield: 60% Fp. 54 . . . 64° C.

EXAMPLE 2 Preparation of O-(2-[¹⁸F]fluoroethyl-L-tyrosine)

The ¹⁸F-containing water was evaporated in the presence of 40 μmoltetra-n-butylammonium-hydrogen carbonate to dryness. Thereafter asolution of 25 mg (37 μmol) N-trityl-O-(2-tosyloxyethyl)-L-tyrosinetert-butyl ester in 0.5 ml acetonitrile was added and the solutionheated to boiling for 5 minutes. Under reduced pressure the solvent wasevaporated, at a temperature of approximately 20° C. 1 ml of a mixtureof trifluoracetic acid/triethylsilane/dichloromethane (1/0,4/2) (v/v/v)was added and stirred for 10 minutes. After dilution with 9 mldichloromethane the solution was led through a silica gel- andsubsequently an aluminum oxide cartridge (1 g each). The cartridges werewashed with 10 ml of a solvent mixture consisting ofn-pentane/diethylether (1/1). The remaining solvent was evaporatedthrough a stream of inert gas, the [¹⁸F]FET raw product was eluted withthe help of a 35 mmol trisodium phosphate solution and purified throughreversed phase High Pressure Liquid Chromatography. The [¹⁸F]FET productfraction was led through a cation exchanger (LiCnrolut SCX, H⁺-form, 1g), thereafter washed with sterile water and the ¹⁸F-labelled amino acideluted with a sodium phosphate buffer of physiologic concentration andthe eluate sterile filtrated.

1. L-Tyrosine derivative of the formula (1)

whereby R¹ is a protective group far the carboxy group selected from thegroup consisting of a methylthiomethyl group, a tetrahydrofuranyl group,a diphenylmethyl group, a para-methoxybenzyl group, a piperonyl group ora tert-butyl group, R²is a protective group for the amino grouprepresented by an arylalkyl group and R³ represents a suitable leavinggroup represented by p-toluenesulfonyloxy, methanesulfonyloxy,trifluoromethanesulfonyloxy or bromine.
 2. The tyrosine derivateaccording to claim 1, whereby R¹ represents a tert-butyl group, R² atriphenylmethyl group and R³ a toluenesulfonyloxy-group.
 3. A method forthe preparation of O-(2-[¹⁸]fluoroethyl)-L-tyrosine whereby an initialcompound of the formula (2)

is converted with R²X to a compound of the formula (3)

and the compound of the formula (3) is converted in a second step into acompound of the formula (1) through the reaction of the hydroxyl groupwith a 1,2-bifunctional ethylene derivative.

and the compound of the formula (1) reacts in a third step with atetraalkylammonium[18F]fluoride of the formulaR₄N⁺¹⁸F* or a cryptate of the formula[K⊂2.2.2]¹⁸F in the presence of a phase transfer catalyst, in an aproticsolvent, to the compound of the formula (5)

and the groups R¹ and R² are split off in a separate step in an acidicsystem and the resulting product O-(2-[18F]fluoroethyl-L-tyrosine) isthereafter purified, whereby R¹, R² and R³ have the same meaning as inclaim 1, X represents halogen and R represents an alkyl group with 3 to6 carbon atoms.
 4. The method according to claim 3, whereby R is ann-butyl group.
 5. The method according to claim 3, whereby the phasetransfer catalyst is tetrabutylammonium hydrogen carbonate.
 6. Themethod according to any of claims 3 to 5, whereby the aprotic solvent isselected from the group consisting of acetonitrile, N,N-dimethylacetamide, N,N-dimethyl formamide, dimethyl sulfoxide andhexamethylphosphoric triamide.
 7. The method according to any of claims3 to 5, whereby the acidic system in the last step of the methodcomprises trifluoroacetic acid in dichloromethane.
 8. The methodaccording to claim 6, whereby the acidic system in the last step of themethod comprises trifluoroacetic acid in dichloromethane.